Compositions and methods for inhibition of HIV-1 infection

ABSTRACT

This invention provides a composition which comprises an admixture of two compounds, wherein one compound retards attachment of HIV-1 to a CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell and the other compound retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate, wherein the relative mass ratio of the compounds in the admixture ranges from about 100:1 to about 1:100, the composition being effective to inhibit HIV-1 infection of the CD4+ cell. This invention also provides a method of inhibiting HIV-1 infection of a CD4+ cell which comprises contacting the CD4+ cell with an amount of the above composition effective to inhibit HIV-1 infection of the CD4+ cell so as to thereby inhibit HIV-1 infection of the CD4+ cell.

COMPOSITIONS AND METHODS FOR INHIBITION OF HIV-1 INFECTION

[0001] Throughout this application, various publications are referencedwithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citations for these references may be foundimmediately preceding the claims.

BACKGROUND OF THE INVENTION

[0002] Infection of cells by human immunodeficiency virus type 1 (HIV-1)is mediated by the viral envelope (env) glycoproteins gp120 and gp41,which are expressed as a noncovalent, oligomeric complex on the surfaceof virus and virally infected cells. HIV-1 entry into target cellsproceeds at the cell surface through a cascade of events that include(1) binding of the viral surface glycoprotein gp120 to cell surface CD4,which is the primary receptor for HIV-1, (2) env binding to fusioncoreceptors such as CCR5 and CXCR4, and (3) multiple conformationalchanges in gp41. During fusion, gp41 adopts transient conformations thatinclude a prehairpin fusion intermediate that ultimately folds into aconformation capable of mediating fusion. These events culminate infusion of the viral and cellular membranes and the subsequentintroduction of the viral genome into the target cell. A similarsequence of molecular events is required for infection to spread viafusion of infected and uninfected cells. Each stage of the viral entryprocess can be targeted for therapeutic intervention.

[0003] HIV-1 attachment can be inhibited both by agents that bind theviral envelope glycoproteins and by agents that bind human CD4. Notably,HIV-1 attachment can be inhibited by compounds that incorporate thegp120-binding domains of human CD4 and molecular mimics thereof [1-7].Because this interaction between gp120 and CD4 is essential for virusinfection, CD4-based molecules have the potential to target most if notall strains of HIV-1. In addition, viruses have limited ability todevelop resistance to such molecules.

[0004] The determinants for gp120 binding map to the first extracellulardomain (D1) on CD4 [1], and the amino acids critical for binding centeron a loop comprising amino acids 36-47. Potent HIV-1 inhibitory activityhas been reproduced in a 27-amino acid peptide that mimics this loop andsurrounding structures [7].

[0005] A number of recombinant CD4-based molecules have been developedand tested for clinical activity in man. The first of these containedthe four extracellular domains (D1-D4) of CD4 but lacked thetransmembrane and intracellular regions. This molecule, termed solubleCD4 (sCD4), demonstrated excellent tolerability when administered tohumans at doses ranging to 10 mg/kg [8,9]. Transient reductions inplasma levels of infectious HIV-1 were observed in certain patientstreated with sCD4. The short half-life of sCD4 in humans (45 minutesfollowing intravenous administration) was identified as one obstacle tousing this agent for chronic therapy.

[0006] Second-generation CD4-based proteins were developed withincreased serum half-life. These CD4-immunoglobulin fusion proteinscomprised the D1D2 domains of CD4 genetically fused to the hinge CH2 andCH3 regions of human IgG molecules. These divalent proteins derive HIV-1neutralizing capacity from their CD4 domains and Fc effector functionsfrom the IgG molecule. A CD4-IgG1 fusion protein was shown to haveexcellent tolerability and improved pharmacokinetics in Phase I clinicaltesting [10]. The antiviral evaluations were inconclusive.

[0007] More recently, a third-generation tetravalent CD4-IgG2 fusionprotein was developed that comprises the D1D2 domains of CD4 geneticallyfused to the heavy and light chain constant regions of human IgG2. Thisagent binds the HIV-1 envelope glycoprotein gp120 with nanomolaraffinity [5] and may inhibit virus attachment both by receptor blockadeand by detaching gp120 from the virion surface, thereby irreversiblyinactivating the virus. In standard PBMC-based neutralization assays,CD4-IgG2 neutralized primary HIV-1 isolates derived from all majorsubtypes and outlier groups. The CD4-IgG2 concentrations required toachieve a 90% reduction in viral infectivity, the in vitro IC90, wereapproximately 15-20 μg/ml [11], concentrations that are readilyachievable in vivo. CD4-IgG2 was similarly effective in neutralizingHIV-1 obtained directly from the plasma of seropositive donors in an exvivo assay, indicating that this agent is active against the diverseviral quasispecies that are encountered clinically [12]. CD4-IgG2 alsoprovided protection against infection by primary isolates in thehu-PBL-SCID mouse model of HIV-1 infection [13]. Recent analyses havedemonstrated that CD4-IgG2's ability to neutralize primary viruses isindependent of their coreceptor usage [14].

[0008] Compared with mono- or divalent CD4-based proteins, CD4-IgG2 hasconsistently demonstrated as much as 100-fold greater potency atinhibiting primary HIV-1 isolates [5,12,14,15]. The heightened potencymay derive from CD4-IgG2's ability to bind virions with increasedvalency/avidity and its steric juxtaposition of two gp120 binding siteson each Fab-like arm of the immunoglobulin molecule. The larger Fab-likearms of CD4-IgG2 are also more likely to span HIV-1 envelope spikes onthe virion. In a variety of preclinical models, CD4-IgG2's anti-HIV-1activity has been shown to compare favorably with those of the rarehuman monoclonal antibodies that broadly and potently neutralize primaryHIV-1 isolates [5,11,14,15]. In addition, CD4-IgG2 therapy is inprinciple less susceptible to the development of drug-resistant virusesthan therapies employing anti-env monoclonal antibodies or portions ofthe highly mutable HIV-1 envelope glycoproteins. These propertiessuggest that CD4-IgG2 may have clinical utility as an agent thatneutralizes cell-free virus before it has the opportunity to establishnew rounds of infection. In addition to treatment, CD4-IgG2 may haveutility in preventing infection resulting from occupational, perinatalor other exposure to HIV-1.

[0009] In Phase I clinical testing, single-dose CD4-IgG2 demonstratedexcellent pharmacology and tolerability. In addition, measurableantiviral activity was observed by each of two measures. First, astatistically significant acute reduction in plasma HIV RNA was observedfollowing administration of a single 10 mg/kg dose. In addition,sustained reductions in plasma levels of infectious HIV were observed ineach of two patients tested. Taken together, these observations indicatethat CD4-IgG2 possesses antiviral activity in humans [16].

[0010] In addition to CD4-based proteins and molecular mimics thereof,HIV-1 attachment can also be inhibited by antibodies and nonpeptidylmolecules. Known inhibitors include (1) anti-env antibodies such asIgG1b12 and F105 [17,18], (2) anti-CD4 antibodies such as OKT4A, Leu 3a,and humanized versions thereof [19,20], and (3) nonpeptidyl agents thattarget either gp120 or CD4 [21], [22-24]. The latter group of compoundsincludes aurintricarboxylic acids, polyhydroxycarboxylates, sulfonicacid polymers, and dextran sulfates.

[0011] Several agents have been identified that block HIV-1 infection bytargeting gp41 fusion intermediates. These inhibitors may interact withthe fusion intermediates and prevent them from folding into finalfusogenic conformations. The first such agents to be identifiedcomprised synthetic or recombinant peptides corresponding to portions ofthe gp41 ectodomain predicted to form hydrophobic alpha helices. Onesuch region is present in both the amino and carboxy segments of theextracellular portion of gp41, and recent crystallographic evidencesuggests that these regions interact in the presumed fusogenicconformation of gp41 [25,26]. HIV-1 infection can be inhibited by agentsthat bind to either N- or C-terminal gp41 epitopes that are exposedduring fusion. These agents include the gp41-based peptides T-20(formerly known as DP178), T-1249, DP107, N34, C28, and various fusionproteins and analogues thereof [27-33]. Other studies have identifiedinhibitors that comprise non-natural D-peptides and nonpeptidyl moieties[34,35]. Clinical proof-of-concept for this class of inhibitors has beenprovided by T-20, which reduced plasma HIV RNA levels by as much as 2logs in Phase I/II human clinical testing [36]. The broad antiviralactivity demonstrated for this class of inhibitors reflects the highdegree of gp41 sequence conservation amongst diverse strains of HIV-1.

[0012] Recent studies [37] have demonstrated the possibility of raisingantibodies against HIV-1 fusion intermediates. This work employed“fusion-competent” HIV vaccine immunogens that capture transient fusionintermediates formed upon interaction of gp120/gp41 with CD4 and fusioncoreceptors. The immunogens used in these studies were formalin-fixedcocultures of cells that express HIV-1 gp120/gp41 and cells that expresshuman CD4 and CCR5 but not CXCR4. The antibodies elicited by thevaccines demonstrated unprecedented breadth and potency in inhibitingprimary HIV-1 isolates regardless of their coreceptor usage, indicatingthat the antibodies were raised against structures such as gp4l fusionintermediates that are highly conserved and transiently exposed duringHIV-1 entry. This class of antibodies does not include the anti-gp41monoclonal antibody known as 2F5, which interacts with an epitope thatis constitutively presented on virus particles prior to fusion [38].

[0013] Previously, synergistic inhibition of HIV-1 entry has beendemonstrated using certain anti-env antibodies used in combination withother anti-env antibodies [39-44], anti-CD4 antibodies [45], orCD4-based proteins [6]. Similarly, synergies have been observed usinganti-CCR5 antibodies used in combination with other anti-CCR5antibodies, CC-chemokines, or CD4-based proteins [46]. However, no priorstudies have examined the potential synergistic effects of combininginhibitors of gp41 fusion intermediates with inhibitors of other stagesof HIV-1 entry. In particular, no studies have examined combinations ofinhibitors of gp41 fusion intermediates and HIV-1 attachment.

SUMMARY OF THE INVENTION

[0014] This invention provIdes a composition which comprises anadmixture of two compounds, wherein one compound retards attachment ofHIV-1 to a CD4+ cell by retarding binding of HIV-1 gp120 envelopeglycoprotein to CD4 on the surface of the CD4+ cell and the othercompound retards gp41 from adopting a conformation capable of mediatingfusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope ona gp41 fusion intermediate, wherein the relative mass ratio of thecompounds in the admixture ranges from about 100:1 to about 1:100, thecomposition being effective to inhibit HIV-1 infection of the CD4+ cell.

[0015] This invention provides a method of inhibiting HIV-1 infection ofa CD4+ cell which comprises contacting the CD4+ cell with an amount ofthe above composition effective to inhibit HIV-1 infection of the CD4+cell so as to thereby inhibit HIV-1 infection of the CD4+ cell.

[0016] This invention provides a method of inhibiting HIV-1 infection ofa CD4+ cell which comprises contacting the CD4+ cell with an amount of acompound which retards attachment of HIV-1 to the CD4+ cell by retardingbinding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface ofthe CD4+ cell effective to inhibit HIV-1 infection of the CD4+ cell andan amount of a compound which retards gp41 from adopting a conformationcapable of mediating fusion of HIV-1 to a CD4+ cell by bindingnoncovalently to an epitope on a gp41 fusion intermediate so as tothereby inhibit HIV-1 infection of the CD4+ cell.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1

[0018] Synergistic inhibition of HIV-1 entry CD4-IgG2 (--▪--), T-20(----), and a 25:1 CD4-IgG2: T-20 combination (...▴...) were analyzedfor inhibition of HIV-1 entry in an env-mediated membrane fusion (RET)assay. Inhibitors were added to a mix of HeLa-Env_(JR-FL) ⁺ and PM1cells previously labeled with F18 and R18 respectively. Fluorescence RETwas measured after 4h of incubation, and percent inhibition wascalculated as described [19]. Results are mean values from threeindependent experiments. The data were analyzed according to the medianeffect principle described in Equation (1). The best-fit parameters forK and m are 0.31 μg/ml and 0.73 for CD4-IgG2, 0.017 μg/ml and 0.92 forT-20, and 0.11 μg/ml and 1.0 for their combination. These curves areplotted and indicate a reasonable goodness-of-fit between experiment andtheory (r²=0.983, 0.998, and 0.996 for CD4-IgG2, T-20, and theircombination, respectively). To normalize for the differences inpotencies of the compounds, separate concentrations scales are used forCD4-IgG2 and the 25:1 CD4-IgG2:T-20 mixture and for T-20, as indicated.

[0019]FIG. 2

[0020] Combination indices for inhibition of HIV-1 entry by combinationsof CD4-IgG2 and T-20. CD4-IgG2, T-20 and fixed mass ratios thereof wereanalyzed in the RET assay for the ability to inhibit env-mediatedmembrane fusion. The 25:1 (high) combination examined 10 three-foldserial dilutions of 250 μg/ml CD4-IgG2, 10 μg/ml T-20 and theircombination. The 25:1 (low) combination examined 10 three-fold serialdilutions of 50 μg/ml CD4-IgG2, 2 μg/ml T-20 and their combination. The5:1 combination examined 10 three-fold serial dilutions of 50 μg/mlCD4-IgG2, 2 μg/ml T-20, and their combination. The 1:1 combinationexamined 10 three-fold serial dilutions of 10 μg/ml CD4-IgG2, 10 μg/mlT-20 and their combination. Inhibition data from three or moreindependent assays were averaged prior to analysis. Dose-response curvesfor the various inhibitors and combinations were fit to Equation (1),which was then rearranged to calculate the inhibitor concentrationsrequired to effect a given percent inhibition. The concentrations of theindividual agents in an inhibitory mixture were calculated from theirknown mass ratios. These values were then used to calculate theCombination Index (CI) according to Equation (2). CI<1 indicatessynergy, CI=1 indicates additive effects, and CI>1 indicates antagonism.

[0021]FIG. 3

[0022] Dose reductions observed for synergistic combinations of CD4-IgG2and T-20. CD4-IgG2, T-20 and a 25:1 fixed mass ratio thereof were testedin the RET assay for the ability to inhibit env-mediated membranefusion. Inhibition data from six independent assays were averaged. K andm were determined by curve-fitting the dose-response curves, andEquation (1) was rearranged to allow calculation of c for a given f forthe single agents and their combination. Dose Reduction is the ratio ofthe inhibitor concentrations required to achieve a given degree ofinhibition when the inhibitor is used alone v. in a synergisticcombination.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The plasmids CD4-IgG2-HC-pRcCMV and CD4-kLC-pRcCMV were depositedpursuant to, and in satisfaction of, the requirements of the BudapestTreaty on the International Recognition of the Deposit of Microorganisms(the “Budapest Treaty”) for the Purposes of Patent Procedure with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 under ATCC Accession Nos. 75193 and 75194,respectively. The plasmids were deposited with ATCC on Jan. 30, 1992.The plasmid designated pMA243 was similarly deposited in accordance withthe Budapest Treaty with ATCC under Accession No. 75626 on Dec. 16,1993.

[0024] As used herein, the following standard abbreviations are usedthroughout the specification to indicate specific amino acids: A = ala =alanine R = arg = arginine N = asn = asparagine D = asp = aspartic acidC = cys = cysteine Q = gln = glutamine E = glu = glutamic acid G = gly =glycine H = his = histidine I = ile = isoleucine L = leu = leucine K =lys = lysine M = met = methionine F = phe = phenylalanine P = pro =proline S = ser = serine T = thr = threonine W = trp = tryptophan Y =tyr = tyrosine V = val = valine

[0025] This invention provides a composition which comprises anadmixture of two compounds, wherein one compound retards attachment ofHIV-1 to a CD4+ cell by retarding binding of HIV-1 gp120 envelopeglycoprotein to CD4 on the surface of the CD4+ cell and the othercompound retards gp41 from adopting a conformation capable of mediatingfusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope ona gp41 fusion intermediate, wherein the relative mass ratio of thecompounds in the admixture ranges from about 100:1 to about 1:100, thecomposition being effective to inhibit HIV-1 infection of the CD4+ cell.

[0026] As used herein, “HIV-1” means the human immunodeficiency virustype-1. HIV-1 includes but is not limited to extracellular virusparticles and the forms of HIV-1 associated with HIV-1 infected cells.HIV-1_(JR-FL) is a strain that was originally isolated at autopsy fromthe brain tissue of an AIDS patient [47]. The virus was co-cultured withlectin-stimulated normal human peripheral blood mononuclear cells. Thevirus has been cloned and the DNA sequences of its envelopeglycoproteins are known (Genbank Accession #U63632). In terms ofsensitivity to inhibitors of viral entry, HIV-1_(JR-FL) is known to behighly representative of primary HIV-1 isolates [11,14,15,48-50].

[0027] As used herein, “gp41 fusion intermediates” includes structures,conformations, and oligomeric states that are preferentially andtransiently presented or exposed on the HIV-1 envelope glycoprotein gp41during the process of HIV-1 env-mediated membrane fusion. Theseintermediates may form upon interaction of HIV-1 with cellular receptorsor may be present in partially or fully occluded states on HIV-1 priorto its interaction with cellular receptors. “gp41 fusion intermediates”do not include fusogenic gp41 conformations that cannot provide targetsfor therapeutic intervention.

[0028] The gp41 fusion intermediates may contain multiple epitopes thatare transiently exposed during fusion and can provide targets fortherapeutic intervention. As used herein, an “N-terminal gp41 epitope”may comprise all or portions of the sequences from amino acid A541 toQ590. As used herein, a “C-terminal gp41 epitope” may comprise all orportions of the sequences from amino acid W628 to L663. These epitopeshave the potential to form coiled-coils of interacting alpha helicalsegments by virtue of heptad (sequence of seven amino acids) repeatscontaining hydrophobic amino acids at positions 1 and 4 of the heptad.The amino acid numbering system is for the HxB2 isolate of HIV-1(Genbank Protein Accession No. AAB50262). Because of the sequencevariability of HIV-1 envelope proteins, the composition, size andprecise location of such sequences may be different for different viralisolates. The gp41 fusion intermediates may also present other linear orconformational epitopes that are transiently expressed during HIV-1entry. An inhibitor may target multiple epitopes present on gp41 fusionintermediates. Alternatively, separate inhibitors may be used incombination to target one or more epitopes present on gp41 fusionintermediates.

[0029] As used herein, “fusogenic” means capable of mediating membranefusion. As used herein, “HIV-1 fusion coreceptor” means a cellularreceptor that mediates fusion between the target cell expressing thereceptor and HIV-1 or an HIV-1 envelope glycoprotein⁺ cell. HIV-1 fusionco-receptors include but are not limited to CCR5, CXCR4 and otherchemokine receptors. As used herein, “fusion” means the joining or unionof the lipid bilayer membranes found on mammalian cells or viruses suchas HIV-1. This process is distinguished from the attachment of HIV-1 toa target cell. Attachment is mediated by the binding of the HIV-1exterior glycoprotein to the human CD4 receptor, which is not a fusionco-receptor.

[0030] As used herein, “retards” means that the amount is reduced. Asused herein, “attachment” means the process that is mediated by thebinding of the HIV-1 envelope glycoprotein to the human CD4 receptor,which is not a fusion co-receptor. As used herein, “CD4” means themature, native, membrane-bound CD4 protein comprising a cytoplasmicdomain, a hydrophobic transmembrane domain, and an extracellular domainwhich binds to the HIV-1 gp120 envelope glycoprotein.

[0031] As used herein, “epitope” means a portion of a molecule ormolecules that form a surface for binding antibodies or other compounds.The epitope may comprise contiguous or noncontiguous amino acids,carbohydrate or other nonpeptidyl moities or oligomer-specific surfaces.

[0032] The compounds of the subject invention have shown to demonstratea synergistic effect. As used herein, “synergistic” means that thecombined effect of the compounds when used in combination is greaterthan their additive effects when used individually.

[0033] In one embodiment of the above composition, the compound whichretards attachment of HIV-1 to the CD4+ cell by retarding binding ofHIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cellis a CD4-based protein. As used herein, “CD4-based protein” means anyprotein comprising at least one sequence of amino acid residuescorresponding to that portion of CD4 which is required for CD4 to form acomplex with the HIV-1 gp120 envelope glycoprotein.

[0034] In one embodiment, the CD4-based protein is a CD4-immunoglobulinfusion protein. In one embodiment, the CD4-immunoglobulin fusion proteinis CD4-IgG2, which is a heterotetrameric CD4-human IgG2 fusion protein.In one embodiment, the CD4-IgG2 comprises two heavy chains and twolights chains, wherein the heavy chains are encoded by an expressionvector designated CD4-IgG2HC-pRcCMV (ATCC Accession No. 75193) and thelight chains are encoded by an expression vector designatedCD4-kLC-pRcCMV (ATCC Accession No. 75194).

[0035] In one embodiment of the above composition, the compound whichretards attachment of HIV-1 to the CD4+ cell by retarding binding ofHIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cellis a protein, the amino acid sequence of which comprises that of aprotein found in HIV-1 as an envelope glycoprotein. In one embodiment,the protein binds to an epitope of CD4 on the surface of the CD4+ cell.The envelope glycoprotein includes but is not limited to gp120, gp160,or gp140.

[0036] In one embodiment of the above composition, the compound whichretards the attachment of HIV-1 to the CD4+ cell by retarding binding ofHIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cellis an antibody or portion of an antibody. In one embodiment, theantibody is a monoclonal antibody. The antibody of the subject inventionmay be a human, humanized or chimeric antibody. In one embodiment, theportion of the antibody is a Fab fragment of the antibody. In oneembodiment, the portion of the antibody comprises the variable domain ofthe antibody. In one embodiment, the portion of the antibody comprises aCDR portion of the antibody. The monoclonal antibody of the subjectinvention includes but is not limited to an IgG, IgM, IgD, IgA, or IgEmonoclonal antibody.

[0037] As used herein, “antibody” means an immunoglobulin moleculecomprising two heavy chains and two light chains and which recognizes anantigen. The immunoglobulin molecule may derive from any of the commonlyknown classes, including but not limited to IgA, secretory IgA, IgG andIgM. IgG subclasses are also weld known to those in the art and includebut are not limited to human IgG1, IgG2, IgG3 and IgG4. It includes, byway of example, both naturally occurring and non-naturally occurringantibodies. Specifically, “antibody” includes polyclonal and monoclonalantibodies, and monovalent and divalent fragments thereof. Furthermore,“antibody” includes chimeric antibodies, wholly synthetic antibodies,single chain antibodies, and fragments thereof. The antibody may be ahuman or nonhuman antibody. A nonhuman antibody may be humanized byrecombinant methods to reduce its immunogenicity in man. Methods forhumanizing antibodies are known to those skilled in the art.

[0038] In one embodiment, the monoclonal antibody binds to an HIV-1envelope glycoprotein. The HIV-1 envelope glycoprotein includes but isnot limited to gp120 and gp160.

[0039] In one embodiment, the HIV-1 envelope glycoprotein is gp120 andthe monoclonal antibody which binds to gp120 is IgG1b12 or F105. IgG1b12is listed as item #2640 in the NIH AIDS Research and Reference ReagentProgram Catalog. F105 is listed as item #857 in the NIH AIDS Researchand Reference Reagent Program Catalog.

[0040] In one embodiment, the antibody binds to an epitope of CD4 on thesurface of the CD4+ cell.

[0041] In one embodiment, the compound which retards attachment of HIV-1to the CD4+ cell by retarding binding of HIV-1 gp120 envelopeglycoprotein to CD4 on the surface of the CD4+ cell is a peptide. In oneembodiment, the compound which retards attachment of HIV-1 to the CD4+cell is a nonpeptidyl agent. As used herein, “nonpeptidyl” means thatthe agent does not consist in its entirety of a linear sequence of aminoacids linked by peptide bonds. A nonpeptidyl agent may, however, containone or more peptide bonds.

[0042] In one embodiment of the above composition, the compound whichretards gp41 from adopting a conformation capable of mediating fusion ofHIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41fusion intermediate is an antibody.

[0043] In one embodiment, the antibody is a monoclonal antibody. Inanother embodiment, the antibody is a polyclonal antibody.

[0044] In one embodiment of the above composition, the compound whichretards gp41 from adopting a conformation capable of mediating fusion ofHIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41fusion intermediate is a peptide.

[0045] In one embodiment, the compound which retards gp41 from adoptinga conformation capable of mediating fusion of HIV-1 to a CD4+ cell bybinding noncovalently to an epitope on a gp41 fusion intermediate is afusion protein which comprises a peptide selected from the groupconsisting of T-20 (SEQ ID NO: 1), DP107 (SEQ ID NO: 2), N34 (SEQ ID NO:3), C28 (SEQ ID NO: 4), and N34(L6)C28 (SEQ ID NO: 5).

[0046] As used herein, “T-20” and “DP178” are used interchangeably todenote a peptide having the following amino acid sequence:YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:1) and as described [29,32].

[0047] DP+B107 has the following amino acid sequence: (SEQ ID NO:2)NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ

[0048] N34 has the following amino acid sequence:SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR (SEQ ID NO:3)

[0049] C28 has the following amino acid sequence:WMEWDREINNYTSLIHSLIEESQNQQEK (SEQ ID NO:4)

[0050] N34(L6)C28 has the following amino acid sequence: (SEQ ID NO:5)SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARSGGRGGWMEWDREINN YTSLIHSLIEESQNQQEK

[0051] In one embodiment of the above composition, the peptide isselected from the group consisting of T-20 (SEQ ID NO: 1), DP107 (SEQ IDNO: 2), N34 (SEQ ID NO: 3), C28 (SEQ ID NO: 4), and N34(L6)C28 (SEQ IDNO: 5).

[0052] In one embodiment of the above composition, the peptide is T-20(SEQ ID NO: 1).

[0053] In one embodiment of the above composition, the peptide is amutant peptide which (1) consists of amino acids having a sequenceidentical to that of a wildtype peptide selected from the groupconsisting of T-20 (SEQ ID NO: 1), DP-107 (SEQ ID NO: 2), N34 (SEQ IDNO: 3), C28 (SEQ ID NO: 4), and N34(L6)C28 (SEQ ID NO: 5), except for anaddition of at least one glycine residue to a 5′ end of the peptide, toa 3′ end of the peptide, or to both ends of the peptide and (2) retardsgp41 from adopting a conformation capable of mediating fusion of HIV-1to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusionintermediate.

[0054] In one embodiment, the compound which retards gp41 from adoptinga conformation capable of mediating fusion of HIV-1 to a CD4+ cell bybinding noncovalently to an epitope on a gp41 fusion intermediate is anon-peptidyl agent.

[0055] In one embodiment of the above composition, the relative massratio of each such compound in the admixture ranges from about 25:1 toabout 1:1.

[0056] In one embodiment of the above composition, the mass ratio isabout 25:1.

[0057] In one embodiment of the above composition, the mass ratio isabout 5:1.

[0058] In one embodiment of the above composition, the mass ratio isabout 1:1.

[0059] In one embodiment of the above composition, the composition isadmixed with a carrier. The carrier of the subject invention may be anaerosol, intravenous, oral or topical carrier. Pharmaceuticallyacceptable carriers are well known to those skilled in the art. Suchpharmaceutically acceptable carriers may include but are not limited toaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like.

[0060] This invention provides a method of inhibiting HIV-1 infection ofa CD4+ cell which comprises contacting the CD4+ cell with an amount ofthe above composition effective to inhibit HIV-1 infection of the CD4+cell so as to thereby inhibit HIV-1 infection of the CD4+ cell.

[0061] In one embodiment, the CD4+ cell is present in a subject and thecontacting is effected by administering the composition to the subject.

[0062] As used herein, “subject” includes any animal or artificiallymodified animal capable of becoming HIV-infected. Artificially modifiedanimals include, but are not limited to, SCID mice with human immunesystems. The animals include but are not limited to mice, rats, dogs,cats, guinea pigs, ferrets, rabbits, and primates. In the preferredembodiment, the subject is a human.

[0063] In one embodiment, the effective amount of the compositioncomprises from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight of the subject.

[0064] As used herein, “administering” may be effected or performedusing any of the methods known to one skilled in the art, which includesintralesional, intraperitoneal, intramuscular, subcutaneous,intravenous, liposome mediated delivery, transmucosal, intestinal,topical, nasal, oral, anal, ocular or otic delivery. The compounds maybe administered separately (e.g., by different routes of administration,sites of injection, or dosing schedules) so as to combine insynergistically effective amounts in the subject.

[0065] The dose of the composition of the invention will vary dependingon the subject and upon the particular route of administration used.Dosages can range from 0.1 to 100,000 μg/kg. Based upon the composition,the dose can be delivered continuously, such as by continuous pump, orat periodic intervals. For example, on one or more separate occasions.Desired time intervals of multiple doses of a particular composition canbe determined without undue experimentation by one skilled in the art.

[0066] As used herein, “effective dose” means an amount in sufficientquantities to either treat the subject or prevent the subject frombecoming infected with HIV-1. A person of ordinary skill in the art canperform simple titration experiments to determine what amount isrequired to treat the subject.

[0067] This invention provides a method of inhibiting HIV-1 infection ofa CD4+ cell which comprises contacting the CD4+ cell with an amount of acompound which retards attachment of HIV-1 to the CD4+ cell by retardingbinding of HIV-1 gp¹²⁰ envelope glycoprotein to CD4 on the surface ofthe CD4+ cell effective to inhibit HIV-1 infection of the CD4+ cell andan amount of a compound which retards gp41 from adopting a conformationcapable of mediating fusion of HIV-1 to a CD4+ cell by bindingnoncovalently to an epitope on a gp41 fusion intermediate so as tothereby inhibit HIV-1 infection of the CD4+ cell.

[0068] In one embodiment, the CD4+ cell is present in a subject and thecontacting is effected by administering the compounds to the subject.

[0069] In one embodiment, the compounds are administered to the subjectsimultaneously. In another embodiment, the compounds are administered tothe subject at different times. In one embodiment, the compounds areadministered to the subject by different routes of administration.

[0070] The subject invention has various applications which includes HIVtreatment such as treating a subject who has become afflicted with HIV.As used herein, “afflicted with HIV-1” means that the subject has atleast one cell which has been infected by HIV-1. As used herein,“treating” means either slowing, stopping or reversing the progressionof an HIV-1 disorder. In the preferred embodiment, “treating” meansreversing the progression to the point of eliminating the disorder. Asused herein, “treating” also means the reduction of the number of viralinfections, reduction of the number of infectious viral particles,reduction of the number of virally infected cells, or the ameliorationof symptoms associated with HIV-1. Another application of the subjectinvention is to prevent a subject from contracting HIV. As used herein,“contracting HIV-1” means becoming infected with HIV-1, whose geneticinformation replicates in and/or incorporates into the host cells.Another application of the subject invention is to treat a subject whohas become infected with HIV-1. As used herein, “HIV-1 infection” meansthe introduction of HIV-1 genetic information into a target cell, suchas by fusion of the target cell membrane with HIV-1 or an HIV-1 envelopeglycoprotein⁺ cell. The target cell may be a bodily cell of a subject.In the preferred embodiment, the target cell is a bodily cell from ahuman subject. Another application of the subject invention is toinhibit HIV-1 infection. As used herein, “inhibiting HIV-1 infection”means reducing the amount of HIV-1 genetic information introduced into atarget cell population as compared to the amount that would beintroduced without said composition.

[0071] This invention will be better understood from the ExperimentalDetails that follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claims thatfollow thereafter.

EXPERIMENTAL DETAILS A. Materials and Methods 1) Reagents

[0072] Purified recombinant CD4-IgG2 protein was produced by ProgenicsPharmaceuticals, Inc. from plasmids CD4-IgG2-HC-pRcCMV andCD4-kLC-pRcCMV (ATCC Accession Nos. 75193 and 75194, respectively) asdescribed [5]. HeLa-env cells were prepared by transfecting HeLa cells(ATCC Catalog #CCL-2) with HIV-1 gp120/gp41 env-expressing plasmidpMA243 as described [51]. PM1 cells are available from the NationalInstitutes of Health AIDS Reagent Program (Catalog #3038). The T-20peptide was synthesized using standard solid-phase Fmoc chemistry andpurified and characterized as described [31,32].

2) Inhibition of HIV-1 Env-Mediated Membrane Fusion

[0073] HIV-1 envelope-mediated fusion between HeLa-Env_(JR-FL) and PM1cells was detected using a Resonance Energy Transfer (RET) assay. Equalnumbers (2×104) of fluorescein octadecyl ester (F18)-labeledenvelope-expressing cells and octadecyl rhodamine (R18)-labeled PM1cells were plated in 96-well plates in 15% fetal calf serum in phosphatebuffered saline and incubated for 4 h at 37 (C in the presence ofvarying concentrations of CD4-IgG2, T-20 or combinations thereof.Fluorescence RET was measured with a Cytofluor plate-reader (PerSeptiveBiosystems) and % RET was determined as previously described [19].

3) Quantitative Analysis of the Synergistic, Additive or AntagonisticEffects of Combining the Agents

[0074] HIV-1 inhibition data were analyzed according to the CombinationIndex method of Chou and Talay [52,53]. The data are modeled accordingto the median-effect principle, which can be written

f=1/[1+(K/c)^(m)]  (1)

[0075] where f is the fraction affected/inhibited, c is concentration, Kis the concentration of agent required to produce the median effect, andm is an empirical coefficient describing the shape of the dose-responsecurve. Equation (1) is a generalized form of the equations describingMichaelis-Menton enzyme kinetics, Langmuir adsorption isotherms, andHenderson-Hasselbalch ionization equilibria, for which m=1 in all cases.In the present case, K is equal to the IC₅₀ value. K and m aredetermined by curve-fitting the dose-response curves.

[0076] After the best-fit parameters for K and m are obtained for theexperimental agents and their combination, Equation (1) is rearranged toallow for calculation of c for a given f. The resulting table of values(e.g., Figure X) is used to calculate the Combination Index (CI) usingthe equation

CI=c _(1m) /c ₁ +c _(2m) /c ₂ +c _(1m) c _(2m) /c ₁ c ₂   (2)

[0077] where

[0078] c₁=concentration of compound 1 when used alone

[0079] c₂=concentration of compound 2 when used alone

[0080] C_(1m)=concentration of compound 1 in the mixture

[0081] c_(2m)=concentration of compound 2 in the mixture

[0082] All concentrations are those required to achieve a given degreeof inhibition. Equation (2) is used when the molecules are mutuallynonexclusive, i.e., have different sites of action. Since this is thelikely scenario for inhibitors of HIV-1 attachment and gp41 fusionintermediates, Equation (2) was used for all Combination Indexcalculations. Mutually nonexclusive calculations provide a moreconservative estimate of the degree of synergy that mutually exclusivecalculations, for which the c_(1m)c_(2m)/c₁c₂ term is dropped. CI<1indicates synergy, CI=1 indicates purely additive effects, and CI>1indicates antagonism. In general, CI values are most relevant at thehigher levels of inhibition that are required to achieve a measurableclinical benefit.

B. Results and Discussion

[0083] Combinations of inhibitors of HIV-1 attachment and gp41 fusionintermediates were first tested for the ability to inhibit HIV-1env-mediated membrane fusion in the RET assay. This assay has proven tobe a highly successful model of the HIV-1 entry process. In this assay,env-dependent coreceptor usage patterns and cellular tropisms of theparental viruses are accurately reproduced [19]. Indeed, the assay wasinstrumental in demonstrating that CCR5 functions as a requisite fusioncoreceptor and acts at the level of viral entry [54]. The fusion assayand infectious virus are similarly sensitive to inhibition by metalchelators and agents that target the full complement of viral andcellular receptors [19,46,55].

[0084] Dose-response curves were obtained for the agents usedindividually and in combination in both assays. Data were analyzed usingthe median effect principle [52,53]. The concentrations of single-agentsor their mixtures required to produce a given effect were quantitativelycompared in a term known as the Combination Index (CI). CI>1 indicatesantagonism, CI=1 indicates a purely additive effect, and CI<1 indicatesa synergistic effect wherein the presence of one agent enhances theeffect of another.

[0085] Combinations of CD4-IgG2 and T-20 were observed to be potentlysynergistic in inhibiting env-mediated membrane fusion. FIG. 1illustrates representative dose-response curves obtained in the membranefusion assay for CD4-IgG2, T-20, and combinations of the two. The curvefor the combination is highly displaced towards lower inhibitorconcentrations and provides qualitative evidence that CD4-IgG2 and T-20act in a synergistic manner.

[0086] To quantitatively calculate the degree of synergy observedbetween CD4-IgG2 and T-20, we analyzed the dose-response curvesaccording to the Combination Index method [52,53]. The analysis includeddata obtained at 25:1, 5:1, and 1:1 CD4-IgG2:T-20 mass ratios. At the25:1 mass ratio, both high (0-250 μg/ml CD4-IgG2 and 0-10 μg/ml T-20)and low (0-50 μg/ml CD4-IgG2 and 0-2 μg/ml T-20) concentration rangeswere evaluated. As indicated in FIG. 2, potent synergies were observedover these broad ranges of inhibitor ratios and concentrations, with CIvalues as low as 0.20 under optimal conditions. This degree of synergyis remarkable since CI values of 0.2 are rarely observed forcombinations involving anti-HIV-1 antibodies [41-44], reversetranscriptase inhibitors [56], or protease inhibitors [57]. The observedsynergies indicate that HIV-1 attachment and formation of gp41 fusionintermediates are inter-dependent steps. One possibility is thatattachment inhibitors, when used at suboptimal concentrations, may slowbut not abrogate the binding of gp120 to CD4. In this case, gp41 fusionintermediates may be formed and persist on the virus (or infected cell)for longer periods of time at levels below that required for membranefusion and thus provide better targets for inhibitory agents.

[0087] The observed synergies translate into significant reductions inthe amounts of CD4-IgG2 and T-20 needed for inhibition. These reductionsare illustrated in FIG. 3 for CD4-IgG2 and T-20 used in a 25:1 massratio. By way of example, inhibition of viral entry by 95% requires 0.21μg/ml of T-20 used alone, 19 μg/ml of CD4-IgG2 used alone and 1.14 μg/mlof a combination containing 0.044 μg/ml of T-20 and 1.1 μg/ml ofCD4-IgG2. The combination reduces the respective doses of T-20 andCD4-IgG2 by 5- and 17-fold, respectively. Still greater dose reductionsare observed at higher levels of inhibition.

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1 5 1 36 PRT Artificial Sequence Description of Artificial Sequence T20peptide 1 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn GlnGln 1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp AlaSer Leu 20 25 30 Trp Asn Trp Phe 35 2 38 PRT Artificial SequenceDescription of Artificial Sequence DP107 peptide 2 Asn Asn Leu Leu ArgAla Ile Glu Ala Gln Gln His Leu Leu Gln Leu 1 5 10 15 Thr Val Trp GlyIle Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu 20 25 30 Arg Tyr Leu LysAsp Gln 35 3 34 PRT Artificial Sequence Description of ArtificialSequence N34 peptide 3 Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu ArgAla Ile Glu Ala 1 5 10 15 Gln Gln His Leu Leu Gln Leu Thr Val Trp GlyIle Lys Gln Leu Gln 20 25 30 Ala Arg 4 28 PRT Artificial SequenceDescription of Artificial Sequence C28 peptide 4 Trp Met Glu Trp Asp ArgGlu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10 15 Ser Leu Ile Glu GluSer Gln Asn Gln Gln Glu Lys 20 25 5 68 PRT Artificial SequenceDescription of Artificial Sequence N34(L6)C28 peptide 5 Ser Gly Ile ValGln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala 1 5 10 15 Gln Gln HisLeu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln 20 25 30 Ala Arg SerGly Gly Arg Gly Gly Trp Met Glu Trp Asp Arg Glu Ile 35 40 45 Asn Asn TyrThr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn 50 55 60 Gln Gln GluLys 65

What is claimed:
 1. A composition which comprises an admixture of two compounds, wherein one compound retards attachment of HIV-1 to a CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell and the other compound retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate, wherein the relative mass ratio of the compounds in the admixture ranges from about 100:1 to about 1:100, the composition being effective to inhibit HIV-1 infection of the CD4+ cell.
 2. The composition of claim 1, wherein the compound which retards attachment of HIV-1 to the CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell is a CD4-based protein.
 3. The composition of claim 2, wherein the CD4-based protein is a CD4-immunoglobulin fusion protein.
 4. The composition of claim 3, wherein the CD4-immunoglobulin fusion protein is CD4-IgG2, wherein the CD4-IgG2 comprises two heavy chains and two lights chains, wherein the heavy chains are encoded by an expression vector designated CD4-IgG2HC-pRcCMV (ATCC Accession No. 75193) and the light chains are encoded by an expression vector designated CD4-kLC-pRcCMV (ATCC Accession No. 75194).
 5. The composition of claim 1, wherein the compound which retards attachment of HIV-1 to the CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell is a protein, the amino acid sequence of which comprises that of a protein found in HIV-1 as an-envelope glycoprotein.
 6. The composition of claim 5, wherein the protein binds to an epitope of CD4 on the surface of the CD4+ cell.
 7. The composition of claim 6, wherein the envelope glycoprotein is selected from the group consisting of gp120, gp160, and gp140.
 8. The composition of claim 1, wherein the compound which retards the attachment of HIV-1 to the CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell is an antibody or portion of an antibody.
 9. The composition of claim 8, wherein the antibody is a monoclonal antibody.
 10. The composition of claim 9, wherein the monoclonal antibody is a human, humanized or chimeric antibody.
 11. The composition of claim 8, wherein the portion of the antibody is a Fab fragment of the antibody.
 12. The composition of claim 8, wherein the portion of the antibody comprises the variable domain of the antibody.
 13. The composition of claim 8, wherein the portion of the antibody comprises a CDR portion of the antibody.
 14. The composition of claim 9, wherein the monoclonal antibody is an IgG, IgM, IgD, IgA, or IgE monoclonal antibody.
 15. The composition of claim 9, wherein the monoclonal antibody binds to an HIV-1 envelope glycoprotein.
 16. The composition of claim 15, wherein the HIV-1 envelope glycoprotein is selected from the group consisting of gp120 and gp160.
 17. The composition of claim 16, wherein HIV-1 envelope glycoprotein is gp120 and the monoclonal antibody which binds to gp120 is IgG1b12 or F105.
 18. The composition of claim 8, wherein the antibody binds to an epitope of CD4 on the surface of the CD4+ cell.
 19. The composition of claim 1, wherein the compound which retards attachment of HIV-1 to the CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell is a peptide.
 20. The composition of claim 1, wherein the compound which retards attachment of HIV-1 to the CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell is a nonpeptidyl agent.
 21. The composition of claim 1, wherein the compound which retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate is an antibody.
 22. The composition of claim 21, wherein the antibody is a monoclonal antibody.
 23. The composition of claim 1, wherein the compound which retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate is a peptide.
 24. The composition of claim 1, wherein the compound which retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate is a fusion protein which comprises a peptide selected from the group consisting of T-20 (SEQ ID NO: 1), DP107 (SEQ ID NO: 2), N34 (SEQ ID NO: 3), C28 (SEQ ID NO: 4), and N34(L6)C28 (SEQ ID NO: 5).
 25. The composition of claim 23, wherein the peptide is selected from the group consisting of T-20 (SEQ ID NO: 1), DP107 (SEQ ID NO: 2), N34 (SEQ ID NO: 3), C28 (SEQ ID NO: 4), and N34(L6)C28 (SEQ ID NO: 5).
 26. The composition of claim 23, wherein the peptide is T-20 (SEQ ID NO: 1).
 27. The composition of claim 1, wherein the compound which retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate is a non-peptidyl agent.
 28. The composition of claim 1, wherein the relative mass ratio of each such compound in the admixture ranges from about 25:1 to about 1:1.
 29. The composition of claim 28, wherein the mass ratio is about 25:1.
 30. The composition of claim 28, wherein the mass ratio is about 5:1.
 31. The composition of claim 28, wherein the mass ratio is about 1:1.
 32. The composition of claim 1, wherein the composition is admixed with a carrier.
 33. The composition of claim 32, wherein the carrier is an aerosol, intravenous, oral or topical carrier.
 34. A method of inhibiting HIV-1 infection of a CD4+ cell which comprises contacting the CD4+ cell with an amount of the composition of claim 1 effective to inhibit HIV-1 infection of the CD4+ cell so as to thereby inhibit HIV-1 infection of the CD4+ cell.
 35. The method of claim 34, wherein the CD4+ cell is present in a subject and the contacting is effected by administering the composition to the subject.
 36. The method of claim 33, wherein the effective amount of the composition comprises from about 0.000001 mg/kg body weight to about 100 mg/kg body weight of the subject.
 37. A method of inhibiting HIV-1 infection of a CD4+ cell which comprises contacting the CD4+ cell with an amount of a compound which retards attachment of HIV-1 to the CD4+ cell by retarding binding of HIV-1 gp120 envelope glycoprotein to CD4 on the surface of the CD4+ cell effective to inhibit HIV-1 infection of the CD4+ cell and an amount of a compound which retards gp41 from adopting a conformation capable of mediating fusion of HIV-1 to a CD4+ cell by binding noncovalently to an epitope on a gp41 fusion intermediate so as to thereby inhibit HIV-1 infection of the CD4+ cell.
 38. The method of claim 37, wherein the CD4+ cell is present in a subject and the contacting is effected by administering the compounds to the subject.
 39. The method of claim 38, wherein the compounds are administered to the subject simultaneously.
 40. The method of claim 38, wherein the compounds are administered to the subject at different times.
 41. The method of claim 38, wherein the compounds are administered to the subject by different routes of administration. 